Optimization of HIV-1 NCp7 Inhibitors as Topical Microbicides and Mechanism of Action of S-acyl-2-mercaptobenzamide Thioesters. Our efforts have focused on the evaluation of the therapeutic potential of S-acyl-2- mercaptobenzamide thioester (SAMT) compounds targeting the zincbinding domains of the HIV-1 nucleocapsid protein (NCp7). These structures are an excellent target for the development of new antiretroviral and microbicidal agents because of their structural conservation and broad range of function in the viral life cycle. Our previous work has identified three SAMT compounds that were shown to be virucidal and to inhibit cell-to-cell associated transmission of HIV-1 in co-culture systems. We have investigated the mechanism of action of the three active compounds, demonstrating that they specifically eject zinc from the carboxyl-terminal zinc-binding domain of NCp7 by acyl transfer from the SAMT to a zinc-coordinating cysteine. As the tertiary structure of the two zinc-binding domains is nearly identical, the primary structure of the zinc-binding domains likely plays a role in the specificity of the SAMT reaction. We have continued to investigate the mechanism of action of the SAMTs using site directed mutagenesis to better understand the observed specificity for the C-terminal zinc binding domain. We determined that position x+1 (where x is Cys36) needs to be an aromatic residue for reactivity, a lysine is required at position x+2 for acylation, and a hydrogen bond donor in position x+9 is important for optimal reactivity. Furthermore, we have been able to extend the previous mechanism of action of the thioester compounds to include a secondary S to N intramolecular acyl transfer that occurs after the primary acyl transfer from the thioester to a cysteine side chain in the protein. This S-N acyl transfer is irreversible, as the transferred acyl group is quite stable on the lysine. The transfer of the acyl group neutralizes the charge on the lysine residue, which we have found results in improper metal coordination and folding. Thus, we have been able to propose a more complete mechanism in which the SAMT compounds first dock onto NCp7 by stacking on the tryptophan side chain, orienting the thioester linkage for acyl transfer to Cys36 sulfur. The acyl group then rapidly and irreversibly moves to proximal lysine residues, leading to disruption of the protein fold and loss of NCp7 function. In order to assess if the SAMTs were potential microbicide candidates, three SAMT compounds were tested in a cervical explant model. We found that all three were able to block infection by cell-free virus from migratory cells. Importantly, SAMT treatment resulted in production of non-infectious virus from cervical explants and also inhibited dissemination of HIV-1 by cervical explant migratory cells. Recently, we have tested one thioester, SAMT-247, in the Simian/Human Immunodeficiency Virus (SHIV)/rhesus macaque model of HIV-1 transmission in collaboration with Dr. Cecilia Cheng-Mayer at the Aaron Diamond AIDS Research Center in New York. These studies have been performed using a mixture of pathogenic CXCR4 (X4) SHIVSF33A and CCR5 (R5) SHIVSF162P3 viruses so that the relative proportion of the two viruses in the plasma could be evaluated by real-time PCR. In a pilot study, SAMT-247 protected five of six macaques against transmission of both X4 and R5 SHIV. We are continuing to test new potential NCp7 inhibitor microbicides in this model. Identification of a potentially safe and efficacious single or combination candidate microbicide in non-human primates will lead us to more formal testing in preparation for clinical evaluation.